Apoptosis induced by nitric oxide is associated with nuclear p53 protein expression in cultured osteoarthritic synoviocytes

Abstract

Objective. Nitric oxide (NO) is a free radical molecule endogenously produced by NO synthases that may play a critical role in inflammation. It inhibits cell proliferation and may be involved in the induction of apoptosis in various cellular models. Recently, the presence of apoptotic cells was reported in the synovium of osteoarthritic (OA) patients. The aim of this study was to determine whether synovial fibroblasts are target cells for NO- induced apoptosis. The expression of p53 protein was also studied to evaluate the ability of synovial cells to repair DNA fragmentation. Methods. Synoviocytes from OA patients were treated with two NO donors: sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP). Apoptosis was analysed by transmission electron microscopy. DNA content was evaluated by flow cytometric analysis after propidium iodide staining and recognition of DNA strand break determined by the TUNEL (TdT-mediated dUTP nick end labeling) method. P53 protein expression was studied by immunofluorescence using a monoclonal antibody. Results. After 6 hours, cells treated with NO donors (1.25 mM) showed a cytoplasmic condensation and vacuolization. DNA strand break analysis by the TUNEL method confirmed the presence of a DNA fragmentation after 24 hours of NO treatment. There was also a progressive decrease in the DNA diploid peak in response to NO donors. In parallel, p53 protein, constitutively expressed in cytoplasmic synovial cells, showed markedly increased expression after a 6-hour NO exposure and displayed prominent nuclear staining after 12 hours. Conclusions. This study demonstrates the potential role of NO for the induction of synoviocyte apoptosis in OA. The increased expression of p53 in the nucleus may play a protective role in the control of apoptosis.